Experimental Study On Antitumor Effect And Its Possible Pathway Of Resveratrol On Endometrial Carcinoma Cell Line Ishikawa

Objective To evaluate the inhibitory effect of resveratrol on the proliferation and invasion of human endometrial cancer cells (ishikawa) in vitro, and preliminary explore the possible mechanism by which resveratrol acts as an anticancer agent to provide a new path for clinical treatment.Methods Human endometrial carcinoma cell line ishikawa was cultured and treated with different concentrations (0,25,50,100μmol/L)of resveratrol for 12,24 and 48 hours. The proliferation of ishikawa cells was detected by MTT assay. The invasive ability of ishikawa cells was observed with a Transwell cell culture chamber. Then total RNA of ishikawa cells was extracted after treated with different concentrations(0,50μmol/L) resveratrol for 24 hours. Agilent microRNA microarray technology was used for high-throughput screening of miRNA. The list of different expressed microRNAs was generated from SAM statistical analysis. MiR-181a was a microRNA which down-regulated by resveratrol. Recent researches suggested that miR-181a might play some important roles in the pathology process of endometrial carcinoma. The validation of miR-181a was performed by real-time quantitative PCR. miR-181a targeted the 3’UTR of sirtl mRNA through a miR-181a binding site, ishikawa cells were treated with different concentrations (0,25,50,100μmol/L)of resveratrol for 24h. Then western blot analysis was used to detect the influences on protein expression of sirtl and its downstream stat3, ac-stat3 and p-stat3. Recombinant adenovirus Ad-miR-181a, which was constructed in the previous experiment, was transfeced into ishikawa cells, with or without resveratrol treatment, followed by real-time quantitative PCR to evaluate the mRNA expression of miR-181a and sirtl, as well as western blot analysis to observe the protein expression of sirtl,stat3, ac-stat3,p-stat3,cyclinD1 and MMP9.Results Resveratrol inhibited the proliferation of ishikawa cells in a concentration-dependent manner when cells were cultured for 24 and 48 hours. Resveratrol significantly inhibited the invasion ability in time and concentration-dependent manner. SAM statistical software analysis showed that 28 microRNAs were down-regulated and 23 microRNAs were up-regulated. Of these, we focused on miR-181a and its downstream genes. The expression of sirtl protein in ishikawa cells treated with resveratrol for 24 hours was increased in a dose-dependentmanner as determined by western blot analysis. The expression of ac-stat3 and p-stat3 protein were decreased in a dose-dependentmanner. Meanwhile, the expression of stat3 protein showed no tendency to change. After transfection of Ad-miR-181a, the mRNA expression of miR-181a increased, but the mRNA expression of sirtl was unaffected, which coexisted with down-regulation of sirtl and up-regulation of ac-stat3,cyclinD1,MMP9 protein expression. RT-PCR and western blot showed that both sirtl mRNA and protein levels were increased in resveratrol-treated ishikawa cells after transfection of Ad-miR-181a, which coexisted with down-regulation of miR-181a mRNA and ac-stat3,p-stat3,cyclinD1,MMP9 protein levels.Conclusion This study indicated that resveratrol inhibited the proliferation and invasion of ishikawa cells significantly. Moreover, resveratrol decreased miR-181a expression, and partially reversed the effects of Ad-miR-181a transfection on relative protein expression in ishikawa cells. It suggested that the anti-tumor mechanism of resveratrol might depend on miR-181a/SIRT1/STAT3 pathway.